The proposed research represents our longstanding efforts to understand the structure, mechanisms of promoter recognition, polymerase and promoter activation, and transcription initiation and elongation of two phage N4-coded RNA polymerases (RNAP), vRNAP and N4 RNAPII. These RNAPs belong to the T7-like single-subunit RNAP family that includes mitochondrial RNAPs. In contrast to the archetypal T7 RNAP, the N4-encoded and mitochondrial RNAPs require protein factors throughout the transcription cycle. We recently reported the X-ray crystal structures of the N4 mini-vRNAP, the catalytic component of vRNAP, in the apo-form and hairpin-form promoter bound and transcription initiation complexes. This represents a major breakthrough and provides the foundation for addressing questions on the transcription mechanism of factor-dependent single-subunit RNAPs using multidisciplinary approaches as described below. 1. Functional and structural dissection of the vRNAP polypeptide: The vRNAP apo-form is in an inactive conformation, and undergoes a structural rearrangement that leads to activation upon promoter binding. We will identify the sensor in vRNAP and the signal in the hairpin-form promoter that lead to vRNAP activation using biochemical approaches. In addition, we will solve the structure of mini-vRNAP in the transition from apo- form to binary complex by improving preliminary crystals that diffract to 3.4 resolution. Mini-vRNAP uses a transcription elongation factor, EcoSSB, to displace the RNA product from the DNA template. We will identify the EcoSSB site of interaction in the transcription elongation complex (TEC) using site-specific protein-protein crosslinking followed by site-directed mutagenesis. In contrast to T7 RNAP, mini-vRNAP is refractory to loading the DNA/RNA scaffold for TEC formation. Therefore, we will use structure-based protein engineering and the unique heat plasticity of vRNAP to prepare the mini-vRNAP TEC with DNA/RNA scaffold, and solve its structure with or without a EcoSSB peptide. 2. Elucidate the N4 RNAPII mechanism of promoter recognition: N4 RNAPII uses a transcription factor N4gp2, a single-stranded DNA binding protein, to recognize its promoters, which are similar in position and length to yeast mitochondrial RNAP promoters. Using an in vivo reporter assay, we will identify the N4 RNAPII determinants of promoter recognition by isolating N4 RNAPII derivatives that suppress promoter mutations. We will define the gp2 interaction platform on N4 RNAPII by site-specific protein-protein crosslinking followed by site-directed mutagenesis. We will also probe the role of gp2 in N4 RNAPII transcription elongation. To obtain a structural basis for factor-dependent single-subunit RNAP transcription, we will solve the crystal structures of the N4 RNAPII apo-form, promoter bound and TEC with or without gp2. We have recently identified and additional protein, N4gp1, required for N4 RNAPII transcription. We will determine its localization on the template in vivo, purify gp1 and analyze its effect on N4 RNAP transcription initiation.